The overall goal of this RO1 grant, now entering its 19th year of funding, is to understand fundamental mechanisms involved in acute regulation of insulin secretion by glucose and other metabolic fuels. The relevance of the project is underscored by the fact that both type 1 and type 2 diabetes are diseases of insulin deficiency, albeit with different etiologies. Type 1 diabetes occurs when pancreatic islet 2-cells are destroyed by the host immune system. Type 2 diabetes involves loss of key 2-cell functions such as glucose-stimulated insulin secretion (GSIS), and a gradual loss of 2-cell mass by non-autoimmune mechanisms. A clearer understanding of the factors that control 2-cell function is critical for ultimate success in creation of surrogate cells for insulin replacement therapy in type 1 diabetes, and for development of better drugs for enhancing functional 2-cell mass in type 2 diabetes. We use an interdisciplinary approach to elucidate the molecular and biochemical mechanism of fuel-stimulated insulin secretion, including development of novel 2-cell lines, expression profiling, genetic engineering, and comprehensive metabolic analysis by NMR- and mass spectrometry (MS)-based methods. In the past funding cycle (9 years due to this grant being awarded Merit status), we have used these methods to demonstrate the importance of a pyruvate carboxylase-initiated pyruvate/isocitrate cycle in control of GSIS. We now seek to understand the specific byproducts of this pathway that serve as coupling factors for control of insulin secretion. The specific aims of the proposal are: 1) To complete the map of the pyruvate/isocitrate cycle, and to understand how it regulates GSIS; 2) To investigate the link between de novo guanine nucleotide biosynthesis, pyruvate cycling activity, and insulin secretion; 3) To understand the potential contributions of impaired pyruvate cycling activity and guanine nucleotide biosynthesis to defective GSIS in type 2 diabetes.